The T-cell Transcription Regulation Group applies transcriptional approaches to understanding how molecular signaling influences gene regulation in mammalian cells. We use T-cells and the process of T-cell activation as a model system. The work and progress in the group over the past year can be defined in 3 major categories:I.Profiling of Transcriptional TargetsII.Defining kinetic profiles of factor occupancy in vivo using Promoter ArraysI.Profiling of Transcriptional TargetsThis year we have a developed a transcriptional approach termed Profiling of Transcriptional Targets (PTT) to help better define how molecular signaling events are integrated in the nucleus to influence gene transcription. The first element of this approach is a high-throughput transcriptional reporter assay where reporter constructs containing various promoters or other gene regulatory elements are inserted into human T-cells and then challenged with a variety (16 or more) of combinations of mitogenic agents in the presence or absence of numerous (8 or more) pharmacological agents with known or presumed immuno-modulatory activity. As a result, multiple gene regulatory region of specific genes can be assessed under over one hundred (128) conditions. Since this process generates a large volume of "high dimensional" or multivariate data, we have effectively applied the use of several computational methods well suited to the type and volume of this data. These methods include unsupervised neural networks, hierarchical clustering and principal component analysis. By this method, we have been able to determine relationships between specific gene regulatory elements and promoter in terms of how they respond to different mitogens and pharmacological agents. This approach has enabled us to identify an operational signaling pathway in activated T-cells that responds to the addition of IGF-1 by up-regulating signaling through a PI3-kinase dependent pathway to exert reciprocal control over NF-kappa B and AP-1 transcriptional activity. Phylogenetic footprinting of gene regulatory elements that respond to IGF-1 reveals that potentially many genes that influence immune cell function or potential targets for IGF-1 regulation. Preliminary gene expression studies using focussed cDNA immunoarrays confirm this. This year we have confirmed the IGF-1 functions as true costimulatory molecule to increase T-cell proliferation, cell-mediated killing, and survival for CD3+ cells and have identified a feed-forward transcriptional loop where by IGF-1 controls proliferation by signling to the TPL2 proto-oncogene via NF-kappa B pathways.Recent expansion of this technique to examine the transcriptional targeting of thalidomide analogues and histone deacetylase inhibitors has identified a new class of thalidomide analogs referred to as "redox reactive" thalidomides that show selective killing of human leukemic cells. Animal studies are currently under way. II.Kinetic Profiling of Promoter Occupancy in in vivoWe have adapted the chromatin immunoprecipitation assay (ChIP) for highthroughput analysis by combining it with microarray technology (ChIP to chip). This allows us to profile the kinetic association of transcription factors and other co-regulatory elements with tens to potentially thousands of different promoter regions simultaneously. Using this platform we have identified novel classes of genes that are directely regulated by the transcriptional coactivator p300 and the tumor suppressor/coregulator BRCA1.